Isolate And Identify Kallikrein-binding Protein Receptors And Its Anti-tumor Mechanism

IntroductionTo exert its intracellular functions, some cytokines should bind to the membranereceptors to transduction signal pathways, or transported into cells mediated by theirreceptor, then exerting its biological activity. Kal was first identified as a tissuekallikrein binding protein (KBP) and a unique serine proteinase inhibitor (serpin). Kalhas variety of biological functions, including lowering blood pressure, diastolicblood,anti-inflammatory, anti-oxidation, anti-fibrotic, promote neointimal formation,inhibition of angiogenesis and tumor growth,etc. Heparin sulfate proteoglycans(HSPGs)，low-density lipoprotein receptor-related protein6(LRP6), Kruppel-likefactor4are be considered as Kal’s receptors. HSPGs was involved in theanti-angiogenic effection of Kal; LRP6was involved in the anti-tumor effection ofKal;Kruppel-like factor4was involved in the anti-inflammatory effection of Kal. Toa certain extent, the three receptors are partly found to explain the mechanism ofsome functions of Kal. But Kal is a multifunctional protein, it should be the role ofmultichannel and multi-target results. There are many Kal’s receptors have not beendiscovered.ObjectiveThe aim of this study is to isolate and indentify Kal’s receptors on the cellmembrane and verify whether these receptors mediated Kal’s anti-angiogenic andanti-tumor effect. In order to reveal the molecular mechanisms and multifounction ofKal’s receptor.Methods(1) Preparation of recombinant human Kal (rhKal), using Pull-down,LC-MC/MC,Co-immunoprecipitation, Immunofluorescence methods to isolate andindentify Kal-binding proteins.(2) Using siRNA,antibody blocking techniques to assay the role of Kal-bindingproteins in anti-angiogenic and anti-tumor effects.(3) Using flow cytometry, Western Blotting and other methods to analyze theeffect of membrane-binding proteins on mediating rhKal into cell. (4) Using cell culture methods to analysis the effect of rhKal-binding proteinsinvolved in signaling pathways.(5) Using the subcutaneous xenograft model to analyze the molecular mechanismsof candidate rhKal-binding proteins mediated the anti-angiogenic and anti-tumoractivity of rhKalResults(1) We firstly discovered that nucleolin, integrin β3, matrix metalloproteinase2(MMP2), vascular endothelial growth factor receptor1(VEGFR1), VEGFR2arerhKal-binding proteins.(2) We firstly discovered that nucleolin is an important receptor mediated rhKalinto cell.(3) We firstly discovered that nucleolin mediated rhKal anti-angiogenic molecularmechanisms.(4) We firstly discovered that integrin β3mediated the anti-tumor activity of rhKal.(5) We firstly discovered that the molecular mechanisms of integrinβ3mediated theanti-tumor activity of rhKal is that rhKal inhibiting of integrin β3, focal adhesionkinase (FAK), a steroid hormone receptor coactivator (Src) phosphorylation.Conclusion(1) Nucleolin is an important receptor that mediated rhKal into cell,nucleolinparticipate anti-tumor angiogenesis and anti-tumor activity of rhKal.(2) Integrin β3is an important receptor that mediated rhKal’s anti-tumor activity.